title: A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.
abstract: The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.
authors.affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Sciences, Tottori University, Yonago, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nakayama
authors.forename: Eiji
authors.initials: E
authors.lastname: Nanba
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ohbayashi
mesh_terms: D000818:Animals; D016466:CHO Cells; D002477:Cells / Q000378:metabolism*; D002478:Cells, Cultured; D022222:Chromosomes, Artificial, Human / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000379:methods*; D030781:Organisms, Genetically Modified; D011995:Recombination, Genetic / Q000502:physiology; D019076:Transgenes / Q000235:genetics
chemical_list: D019426:Integrases